The present invention relates to substances having the property of binding to cyclin dependent kinase (cdk), and in particular to substances having this property derived from analysis of fragments of p16 protein. The present invention also relates to pharmaceutical compositions comprising these substances and their use in methods of medical treatment, especially in the treatment of hyperproliferative disorders. The invention also relates to methods and uses of these substances in identifying compounds having related activities.
Phosphorylation of the Rb gene product (pRb) by members of the cyclin dependent kinase (cdk) family is an important step in the cells commitment to undergo mitosis. This step is regulated in the later part of the G1 phase of the cell cycle at what is known as the restriction (R) point (6). The cdks are key regulatory factors through which both positively and negatively acting cell signal transduction factors merge. Mitogenic stimulation induces an active complex between the D-cyclin, and cdk4 or cdk6 that is capable of phosphorylating pRb in late G1. These kinases are also the targets for cell growth inhibitory signals arising from contact inhibition, growth factor starvation or TGF-xcex2. The inhibitory signals can block kinase activity by inducing the production or activity of different members of the two rapidly enlarging families of INK4 and p21/KIP cdk-inhibitors that either interfere directly with the kinases or with the cyclin-kinase complexes (7). The family of INK proteins that have been identified consists of p15, p16, p18 and p19 (20, 22, 23).
However, unlike p21cipl/WAF1, which is indirectly linked to tumour suppression activity through p53 transcriptional stimulation (8), the INK4p16 gene is itself deleted or mutated in a large number of human tumours (9-15). Germ line mutations in INK4p16 have been associated with an increased risk of developing melanoma (9,10). The 156 amino acid product of the INK4p16 gene is known as CDKN2 or p16INK4a (referred to in this application as xe2x80x9cp16xe2x80x9d).
We set out to identify and study the region of p16 that interacts with cyclin dependent kinases such as cdk4 and cdk6, and to investigate applications of these properties, in particular the possibility that the binding of cdks by substances comprising a peptide based on this region of p16 could be used in tumour suppression by inhibiting the phosphorylation of Rb protein.
Small peptides can sometimes be powerful tools to identify regions of proteins involved in protein-protein interactions and biological activity (16-19). In this work, we synthesised a series of overlapping 20 amino acid (aa) peptides that spanned the p16 amino acid sequence, and tested the capacity of each biotinylated peptide to interact with 35S-labelled cdk4 and cdk6 expressed in rabbit reticulocyte lysates.
These experiments identified a 20 amino acid synthetic peptide corresponding to residues 84 to 103 of p16 that interacts with cdk4 and cdk6, and inhibits cdk4-cyclin D1 mediated phosphorlotion of Rb protein in vitro. An alanine substitution series defined amino acid residues important for the cdk4 and cdk6 interaction and for the inhibition of Rb phosphorylation. In this application, residues 84 to 103 of p16 correspond to the sequence sets out in FIG. 1C, i.e. DAAREGFLDTLVVLHRAGAR (SEQ ID NO:1).
Further, when coupled to a small peptide carrier molecule and applied directly to tissue culture medium, the p16-derived peptide blocked cell cycle entrance into S-phase in both serum starved human HaCaT cells and other types of cells that are cycling normally. This was associated with an inhibition of pRb phosphorylation in vivo. These results demonstrate that a p16-derived synthetic peptide coupled to a small carrier molecule can mimic the G1-phase arrest associated with overexpression of full length p16 protein. This provides a route to the restoration of the p16 suppressor gene function in human tumours.
Accordingly, in a first aspect, the present invention provides a substance having the property of binding to cyclin dependent kinase (cdk) comprising:
(i) a peptide including amino acid residues 84 to 103 of full length p16 protein, or an active portion or derivative thereof; or,
(ii) a functional mimetic of the fragment, active portion or derivative;
wherein the substance excludes full length p16, p15, p18 and p19 proteins;
Preferably, the cyclin dependent kinase (cdk) is cdk4 or cdk6. The substance preferably also has the property of inhibiting the phosphorylation of Rb protein which is mediated by a complex formed between cdks and cyclin D. This in turn can be used to block cellular differentiation by preventing the entry of cells into the S-phase. As the substances bind cyclin dependent kinases, they can also be used to prevent the formation of the complex between cdks and cyclin D, having the additional biological effect of increasing cyclin D levels in cells. As well as blocking cdk4 and cdk6 dependent phosphorylation of pRb, the substances described herein could be used to target other cellular substrates, including the pRb family members p107 and p130, or other substances that are targets for cdk4 and cdk6 mediated regulation.
In the present invention, xe2x80x9can active portionxe2x80x9d means a portion of the peptide which is less than the full amino acid sequence of the fragment above, but which retains the property of binding to a cyclin dependent kinase (cdk). Preferably, the peptide also has the property of inhibiting pRb phosphorylation.
In the present invention, a xe2x80x9cderivativexe2x80x9d is a protein modified by varying the amino acid sequence of the protein, e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself. Such derivatives of the natural amino acid sequence may involve insertion, addition, deletion or substitution of one or more amino acids, without fundamentally altering the essential activity of the proteins. As an example, a derivative of peptide 6 in which aspartic acid 92 was substituted for alanine was found to be more potent than the peptide 6 (residues 84 to 103) in binding to cdk4 and cdk6, and having a greater inhibition of pRb phosphorylation. Other derivatives include inserting one or more amino acid residues between amino acid motifs FLD ard LVVL.
In the present invention, xe2x80x9cfunctional mimeticxe2x80x9d means a substance which may not contain a fragment or active portion of the p16 amino acid sequence, and probably is now a peptide at all, but which has some or all of the properties of the p16 fragment, in particular the property of binding to a cyclin dependent kinase and/or inhibiting pRb phosphorylation.
In a preferred embodiment, the peptide includes residues 89 to 97 of full length p16 protein. More preferably, the peptide includes the peptide motif FLD, corresponding to amino acids 90 to 92 of full length p16 protein, and/or the peptide motif LVVL (SEQ ID NO:2), corresponding to amino acids 94 to 97 of full length p16 protein. We have also found that both the D and L isoforms of the peptides share the property of binding to cdk and/or inhibiting pRb phosphorylation.
In a further aspect, the present invention provides compounds comprising any of the above substances coupled to carrier molecules, enabling the compounds to be delivered to cells in vivo. In one embodiment, the carrier molecule is a 16 aa peptide sequence derived from the homeodomain of Antennapedia (e.g. as sold under the name xe2x80x9cPenetratinxe2x80x9d), which can be coupled to one of the above substances via a terminal Cys residue. The xe2x80x9cPenetratinxe2x80x9d molecule and its properties are described in WO 91/18981.
In a further aspect, a substance comprising one of the above peptides can be stabilised by coupling to another peptide sequence. Preferably, this allows the peptide to adopt a conformation more closely resembling that of full length p16, typically having the advantage of increasing the activity of the peptide relative to the uncoupled fragment, e.g. so that the peptide fragment has an activity more closely approaching or surpassing that of full length p16.
In further aspects, the present invention provides pharmaceutical compositions comprising one or more of the above substances and the use of these compositions in methods of medical treatment. In a preferred embodiment, the present invention relates to the use of these substances in the preparation of medicaments for the treatment of hyperproliferative disorders, such as cancer, psoriasis or arteriogenesis. In particular, cancers which are p16 negative or associated with the overexpression of cdks are especially likely to respond well to compositions comprising one or more of the above substances.
Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes. For such administration, a parenterally acceptable aqueous solution may be employed which is pyrogen-free and has suitable pH, isotonicity and stability. Those skilled in the art are well able to prepare suitable solutions. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required. Dosage levels can be determined by the those skilled in the art, taking into account the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington""s Pharmaceutical Sciences, 16th edition, Osol, A. (ed). 1980.
In embodiments in which the substances are proteins, the present invention also provides nucleic acid encoding these proteins. Those skilled in the art can readily construct such nucleic acid sequences from the amino acid sequences disclosed herein, taking account of factors such as codon preference in the host used to express the nucleic acid sequences. In embodiments of the invention in which he protein is coupled to a carrier protein, nucleic acid encoding the carrier protein can be linked to the sequence encoding the peptides and the sequences expressed as a fusion.
In further aspects, the present invention provides vectors incorporating the above nucleic acid and host cells transformed with the vectors.
In a further aspect, the present invention provides the use of any one of the above substances in screening for (i) compounds having one or more of the biological activities of the substances described above or (ii) compounds which are binding partners of one of the substances, e.g. antibodies or complementary peptides specific for p16 or a p16 mimetic. Preferably, the substances are peptide fragments of p16 protein. Examples of screening procedures for mimetics or binding partners include:
(a) immobilising the p16 fragments on a solid support and exposing the support to a library of labelled peptides or other candidate compounds, and detecting the binding of the peptides or candidate compound to the p16 fragments;
(b) using labelled cdks and a library of unlabelled candidate compound or peptides;
(c) other combinations of solid phases substrates and binding measurements;
(d) Western blots using the fragments of pig protein and antibodies raised to the p16 fragments and determining the displacement of the antibodies by candidate compounds;
(e) using yeast two hybrid screens to detect candidate peptides which bind to the p16 peptide or to oligonucleotides derived from the p16 fragments (for a description of yeast two hybrid screens see our earlier application WO 96/14334);
(f) using the fragments of p16 protein and/or candidate compounds in cell systems to determine whether the fragments or candidate compounds inhibit phosphorylation of Rb and/or prevent the cells from cycling;
(g) using the fragments of p16 protein and/or candidate compounds in animal models of tumour growth to determine whether the fragments or candidate compounds prevent the occurrence of tumours, reduce tumour size, inhibit tumour growth and/or inhibit tumour cell migration.
In a further aspect, the present invention provides method of identifying compounds which compete with one of the above substances, the method comprising:
(a) binding a predetermined quantity of the substance which is detectably labelled to a cyclin dependent kinase (cdk);
(b) adding a candidate compound; and,
(c) determining the amount of the labelled compound that remains bound to the cdk or which becomes displaced by the candidate compound
In a further aspect, the present invention provides a method of identifying mimetics of one of the above substances, the method comprising:
(a) immobilising one or more candidate compounds on a solid substrate;
(b) exposing the substrate to a labelled cyclin dependent kinase (cdk);
(c) selecting the candidate compounds that bind to cdk.
In the above aspects, preferably the cyclin dependent kinase is cdk4 or cdk6. Preferably, the substance is a fragment of p16 protein, and more preferably the FLD and LVVL motifs disclosed above. Conveniently, the candidate compounds can be selected from a synthetic combinatorial library.
The present invention may further comprise testing the candidate compound for the property of inhibiting pRb phosphorylation and/or testing the compound for the property of inhibiting the entry of cells into the S-phase.
In a further aspect, the present invention provides the use of a fragment of p16 protein including the amino acid motifs FLD, corresponding to amino acid residues 90 to 92 of full length p16 protein, and/or LVVL, corresponding to amino acid residues 94 to 97 of full length p16 protein in the design of an organic compound which is modelled to resemble the three dimensional structure of said amino acid motifs, the organic compound having the properties of binding to cyclin dependent kinase and/or inhibiting pRb phosphorylation.
The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a xe2x80x9cleadxe2x80x9d compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, eg peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing is generally used to avoid randomly screening large number of molecules for a target property.
There are several steps commonly taken in the design of a mimetic from a compound having a given target property Firstly, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, eg by substituting each residue in turn. These parts or residues constituting the active region of the compound are known as its xe2x80x9cpharmacophorexe2x80x9d.
Once the pharmacophore has been found, its structure is modelled to according its physical properties, eg stereochemistry, bonding, size and/or charge, using data from a range of sources, eg spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process.
In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic.
A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
By way of example, the present invention will now be described in more detail with reference to the accompanying figures. The following examples are provided to illustrate the present invention, and should not be interpreted as limiting the scope of the claims.